This invention relates to β-glucan/mannan preparations and to methods for their preparation. In particular, the invention relates to preparations, including β-(1,3/1,6) glucan and mannan, produced from microorganisms including, but not limited, to yeasts.
“Glucan” is a generic term referring to an oligo- or polysaccharide composed predominantly or wholly of the monosaccharide D-glucose. Glucans are widely distributed in nature, and are particularly important for their role in maintaining the structural integrity of bacterial, yeast, and plant cells. For example, glucan, in combination with other polysaccharides such as mannan and chitin, is responsible for the shape and mechanical strength of the cell wall. Glucans typically accounts for approximately 40% to 50% of the weight of the cell wall in these cells.
As polymers of D-glucose, the D-glucose units may be linked together in a variety of ways. For example, glucans with (1,3), (1,4), (1,6) and (1,2) linkages (glucosidic linkages) are all known. The variety of linkages possible means that glucans are normally highly branched compounds. Many forms are possible as a result of this highly variable manner in which this individual glucose units can be joined as well as the overall steric shape of the parent molecule. A common glucan is β-(1,3)-linked glucopyranose (commonly referred to as β-glucan). Cell walls of several species include β-(1,3)-linked glucopyranose coupled with β-(1,6)-linked glucopyranose. For example, the cell wall of Saccharaomyces cerevisiae is primarily composed of β-linked glucan, which is mainly a backbone of β-(1-3)-linked glucose units, with a minor component of inter and intra molecular branching via β-(1-6)-linkages.
Because of their chemical properties, glucans have found a wide variety of uses in the chemical, food and pharmaceutical industries. For example, they may be useful as viscosity imparting agents, emulsifiers, fibers, films, coating substances, supports for affinity chromatography and gel electrophoresis, in cell culture media, as filter pads, and in cement. They are also widely used as food thickeners and as a source of dietary fiber, and as carriers and coating agents in pharmaceutical products. Glucans have been shown to have immunopharmacological activity in humans and animals. For example, strong immunostimulation and protection against pathogenic microorganisms have been demonstrated in shrimp, fish, poultry, swine, cattle, rabbits, mice, rats and humans. Yeast β-glucans may stimulate the innate (non-specific) immune response of vertebrates and invertebrates via interaction with the Toll-like receptor Dectin-1. Such binding stimulates the production of active oxygen species in macrophages and enhances their phagocytosis and killing of microorganisms. These stimulated immune cells also produce cytokins which can circulate throughout the animal and interact with other immune cells to enhance the immune status of the animal.
The purification of β-glucans from yeast and other organisms has been extensively investigated, and a variety of methods is known. Most of these rely on the insolubility of β-(1-3)-glucan in alkali or in organic solvents. The principal known methods are: (a) high temperature extraction with concentrated sodium hydroxide, followed by high temperature extraction with acid and precipitation with ethanol (see, e.g., Manners, D. J. et al., Biochem. J. 135 19-30 (1973), Jamas, S. et al., U.S. Pat. Nos. 4,810,646, 5,028,703, and 5,250,436). Many of these protocols require preliminary homogenization of the yeast cells, and many require multiple repetition of each extraction steps; (b) extraction of yeast cell wall preparations resulting from autolysis or enzyme degradation of yeast with concentrated phenol:water (1:1) (see, e.g., U.S. Pat. No. 4,138,479 by Truscheit, E. et al.); and (c) extraction with organic solvents such as isopropanol, ethanol, acetone, or methanol either alone or in the presence of alkali (see, e.g., European Patent Application No. 515216). Acid treatment is known to reduce the number of β-(1-6)-linkages in the glucan material, which results in an increase in viscosity.
Mannan is a polymer composed of mannose units. In yeasts, mannan is associated with protein in both the external surface of the yeast cell wall, as a muscigenous polysaccharide, and in the inner cell membrane. It generally accounts for about 20-50% of the dry weight of the cell wall. Mannan is linked to a core-peptide chain as an oligomer or polymer. The complex contains about 5-50% proteins. Oligomeric mannan is bonded directly to serine and threonine, whereas polymeric mannan is bonded to aspargine via N-acetylglucosamine. In the manno-protein complex, the mannose units are linked by α-1,6, α-1,2 and α-1,3-linkages.
Mannan-oligosaccharides (MOS) can be released from yeast cell walls by proteolytic action. The released MOS can effectively bind to bacterial pathogens of the intestinal tract and block their ability to colonize the intestinal tract. For example, E. coli, Salmonella spp. and Vibrio cholera have proteins on their surface (lectins) which bind specifically to the mannose sugar residues of the MOS.
Considering the many uses and applications of glucans, there is a clear need in the art for a method of β-glucan/mannan extraction which avoids the use of high concentrations of alkali or acid and the use of high temperatures, which has improved recovery of glucans and mannans, and which results in a biologically useful preparation.